Neuronal control of experimental colitis occurs via sympathetic intestinal innervation.

BACKGROUND: Vagus nerve stimulation is currently clinically evaluated as a treatment for inflammatory bowel disease. However, the mechanism by which this therapeutic intervention can have an immune-regulatory effect in colitis remains unclear. We determined the effect of intestine-specific vagotomy or intestine-specific sympathectomy of the superior mesenteric nerve (SMN) on dextran sodium sulfate (DSS)-induced colitis in mice. Furthermore, we tested the efficacy of therapeutic SMN stimulation to treat DSS-induced colitis in rats. METHODS: Vagal and SMN fibers were surgically dissected to achieve intestine-specific vagotomy and sympathectomy. Chronic SMN stimulation was achieved by implantation of a cuff electrode. Stimulation was done twice daily for 5 minutes using a biphasic pulse (10 Hz, 200 µA, 2 ms). Disease activity index (DAI) was used as a clinical parameter for colitis severity. Colonic cytokine expression was measured by quantitative PCR and ELISA. KEY RESULTS: Intestine-specific vagotomy had no effect on DSS-induced colitis in mice. However, SMN sympathectomy caused a significantly higher DAI compared to sham-operated mice. Conversely, SMN stimulation led to a significantly improved DAI compared to sham stimulation, although no other parameters of colitis were affected significantly. CONCLUSIONS & INFERENCES: Our results indicate that sympathetic innervation regulates the intestinal immune system as SMN denervation augments, and SMN stimulation ameliorates DSS-induced colitis. Surprisingly, intestine-specific vagal nerve denervation had no effect in DSS-induced colitis.

Balancing the autonomic nervous system to reduce inflammation in rheumatoid arthritis.

Imbalance in the autonomic nervous system (ANS) has been observed in many established chronic autoimmune diseases, including rheumatoid arthritis (RA), which is a prototypic immune-mediated inflammatory disease (IMID). We recently discovered that autonomic dysfunction precedes and predicts arthritis development in subjects at risk of developing seropositive RA. In addition, RA patients with relatively high vagus nerve tone (higher parasympathetic parameters, measured by heart rate variability) respond better to antirheumatic therapies. Together, these data suggest that the ANS may control inflammation in humans. This notion is supported by experimental studies in animal models of RA. We have found that stimulation of the so-called cholinergic anti-inflammatory pathway by efferent electrical vagus nerve stimulation (VNS) or pharmacological activation of the alpha7 subunit of nicotinic acetylcholine receptors (α7nAChR) improves clinical signs and symptoms of arthritis, reduces cytokine production and protects against progressive joint destruction. Conversely, increased arthritis activity was observed in alpha7nAChR knockout mice. These studies together with previous work in animal models of sepsis and other forms of inflammation provided the rationale for an experimental clinical trial in patients with RA. We could for the first time show that an implantable vagus nerve stimulator inhibits peripheral blood cytokine production in humans. VNS significantly inhibited TNF and IL-6 production and improved RA disease severity, even in some patients with therapy-resistant disease. This work strongly supports further studies using a bioelectronic approach to treat RA and other IMIDs.

Autonomic Dysfunction Precedes Development of Rheumatoid Arthritis: A Prospective Cohort Study.

BACKGROUND: Heart rate variability (HRV) is a validated method to establish autonomic nervous system (ANS) activity. Rheumatoid arthritis (RA) is accompanied by ANS imbalance. We hypothesized that ANS dysfunction may precede the development of RA, which would suggest that it plays a role in its etiopathogenesis. METHODS: First, we assessed HRV parameters in supine (resting) and upright (active) position in healthy subjects (HS, n=20), individuals at risk of developing arthritis (AR subjects, n=50) and RA patients (RA, n=20). Next, we measured resting heart rate (RHR), a parasympathetic HRV parameter, in an independent prospective cohort of AR subjects (n=45). We also evaluated expression levels of the parasympathetic nicotinic acetylcholine receptor type 7 (α7nAChR) on circulating monocytes. FINDINGS: Both AR subjects (68 beats per minute (bpm), interquartile range (IQR) 68-73) and RA patients (68bpm, IQR 62-76) had a significantly higher RHR compared to HS (60bpm, IQR 56-63). RHR was significantly higher at baseline in individuals who subsequently developed arthritis. Expression levels of α7nAChR were lower in AR subjects with RHR ≥70bpm compared to those with RHR <70bpm, consistent with reduced activity of the parasympathetic cholinergic anti-inflammatory pathway. INTERPRETATION: These data support the notion that autonomic dysfunction precedes the development of RA.

Cholinergic anti-inflammatory pathway in the non-obese diabetic mouse model.

OBJECTIVE: Activation of the cholinergic anti-inflammatory pathway (CAP) has been shown to reduce inflammation in animal models, while abrogation of the pathway increases inflammation. We investigated whether modulation of CAP influences inflammation in the non-obese diabetic (NOD) mouse model for Sjögren's syndrome and type 1 diabetes. METHODS: The alpha-7 nicotinic acetylcholine receptor (α7nAChR) was stimulated with AR-R17779 or nicotine in NOD mice. In a second study, unilateral cervical vagotomy was performed. α7nAChR expression, focus scores, and salivary flow were evaluated in salivary glands (SG) and insulitis score in the pancreas. Cytokines were measured in serum and SG. RESULTS: α7nAChR was expressed on myoepithelial cells in SG. Monocyte chemotactic protein-1 levels were reduced in SG after AR-R17779 treatment and tumor necrosis factor production was increased in the SG of the vagotomy group compared to controls. Focus score and salivary flow were unaffected. NOD mice developed diabetes more rapidly after vagotomy, but at completion of the study there were no statistically significant differences in number of mice that developed diabetes or in insulitis scores. CONCLUSION: Intervention of the CAP in NOD mice leads to minimal changes in inflammatory cytokines, but did not affect overall inflammation and function of SG or development of diabetes.

Vagus nerve stimulation: a new bioelectronics approach to treat rheumatoid arthritis?

There has been a marked improvement in the treatment of rheumatoid arthritis (RA), but most patients do not achieve disease remission. Therefore, there is still a need for new treatments. By screening an adenoviral short hairpin RNA library, we discovered that knockdown of the nicotinic acetylcholine receptor type 7 (α7nAChR) in RA fibroblast-like synoviocytes results in an increased production of mediators of inflammation and degradation. The α7nAChR is intimately involved in the cholinergic anti-inflammatory pathway (CAP). This led us to study the effects of α7nAChR activation in an animal model of RA, and we could show that this resulted in reduced arthritis activity. Accordingly, stimulation of the CAP by vagus nerve stimulation improved experimental arthritis. Conversely, we found aggravation of arthritis activity after unilateral cervical vagotomy as well as in α7nAChR-knockout mice. Together, these data provided the basis for exploration of vagus nerve stimulation in RA patients as a novel anti-inflammatory approach.

Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue.

Antibodies against adeno-associated viral (AAV) vectors are highly prevalent in humans. Both preclinical and clinical studies showed that antibodies against AAV block transduction even at low titers, particularly when the vector is introduced into the bloodstream. Here we measured the neutralizing antibody (NAb) titer against AAV serotypes 2, 5, 6 and 8 in the serum and matched synovial fluid (SF) from rheumatoid arthritis patients. The titer in the SF was lower than that in the matched plasma samples, indicating a difference in distribution of NAb to AAV depending on the body fluid compartment. This difference was more evident for AAV2, against which higher titers were measured. Of all serotypes, anti-AAV5 antibodies were the least prevalent in both the serum and SF. We next evaluated the impact of B-cell depletion on anti-AAV antibodies in rheumatoid arthritis patients who received one or two courses of the anti-CD20 antibody rituximab as part of their disease management. A drop of NAb titer was observed in a subset of those subjects carrying NAb titers ≤1:1000; however, only in a minority of subjects titers dropped below 1:5. This work provides insights into strategies to overcome the limitation of pre-existing humoral immunity to AAV vectors.

Local delivery of beta interferon using an adeno-associated virus type 5 effectively inhibits adjuvant arthritis in rats.

Beta interferon (IFN-beta) is a cytokine with potent immunomodulatory properties and has been described as a promising therapeutic molecule for the treatment of rheumatoid arthritis (RA). IFN-beta was previously overexpressed intra-articularly using an adenoviral vector in rats with adjuvant arthritis (AA) as a model of RA. This effect was powerful, albeit transient due to the vector chosen. Therefore, in the context of pre-clinical development, a delivery vector optimized for intra-articular gene transfer, recombinant adeno-associated virus type 5 (rAAV5), was selected. To exert an optimal effect, protein production should parallel the course of the disease. For this reason, the gene for IFN-beta was placed under the control of an inflammation-responsive [nuclear factor (NF)-kappaB] promoter. After intra-articular injection of the rAAV5 constructs in rats with AA, local transcription of the transgene and production of the IFN-beta protein was found, leading to a pronounced and sustained effect on paw swelling when the expression was under the control of the NF-kappaB-responsive promoter. Additionally, a significant beneficial effect was observed on proteoglycan depletion and erosions. Thus, intra-articular overexpression of IFN-beta using a rAAV5 vector exhibits potential as an innovative therapy for the treatment of RA.

Inflammation-inducible anti-TNF gene expression mediated by intra-articular injection of serotype 5 adeno-associated virus reduces arthritis.

BACKGROUND: The tumor necrosis factor (TNF)-alpha plays a central role in rheumatoid arthritis (RA) and current biotherapies targeting TNF-alpha have a major impact on RA treatment. The long-term safety concerns associated with the repetitive TNF blockade prompt optimization of therapeutic anti-TNF approaches. Since we recently demonstrated that intra-articular gene transfer using a recombinant adeno-associated virus serotype 5 (rAAV5) efficiently transduces arthritic joints, we evaluate its effect on collagen-induced arthritis (CIA) when encoding TNF antagonists. METHODS: Recombinant AAV5 vectors encoding the human TNFRp55 extracellular domain fused to the Fc region of mice IgG1 (TR1) or a small molecular weight dimeric human TNFRp75 extracellular domain (TR2), under two different promoters, the CMV or a chimeric NF-kappaB-based promoter inducible by inflammation, were injected into mouse CIA joints. RESULTS: Best protection against arthritis was obtained with the rAAV5 encoding the TR1, as reflected by delayed disease onset, decreased incidence and severity of joint damage. This effect was associated with a transient expression of the anti-TNF agent when expressed under a NF-kappaB-responsive promoter, only detectable during disease flare, while the antagonist expression was rapidly increased and stable when expressed from a CMV promoter. Importantly, using the intra-articular administration of the rAAV5-NF-kappaB-TR1 vector, we observed a striking correlation between local TR1 expression and inflammation. CONCLUSIONS: These findings strongly support the feasibility of improving the safety of anti-TNF approaches for the treatment of arthritis by local rAAV5-mediated gene expression under an inflammation-responsive promoter, able to provide a limited, transient and therapeutically relevant expression of anti-TNF compounds.

Reduction of arthritis following intra-articular administration of an adeno-associated virus serotype 5 expressing a disease-inducible TNF-blocking agent.

BACKGROUND: In the context of preclinical development, we studied the potential of intra-articular gene delivery using a recombinant adeno-associated virus 5 (rAAV5) encoding a chimeric human tumour necrosis factoralpha (TNFalpha) soluble receptor I linked to a mouse immunoglobulin heavy chain Fc portion (TNF receptor I; TNFRI-Ig). METHODS: Expression was under control of a nuclear factor kappa B (NFkappaB)-responsive promoter and compared with a cytomegalovirus (CMV) promoter (rAAV5.NFkappaB-TNFRI-Ig and rAAV5.CMV-TNFRI-Ig, respectively). RESULTS: Fibroblast-like synoviocytes transduced in vitro with rAAV5.NFkappaB-TNFRI-Ig were able to produce TNFRI-Ig protein in response to several stimuli, and this was inhibited upon treatment with a specific NFkappaB blocking agent. A bioassay revealed that the synthesised TNFRI-Ig was bioactive, showing a higher affinity for human than for rat TNFalpha. Transcription of the transgene and protein production were detectable in joints injected with both constructs. No dissemination of the vector was observed outside the joints. A significant reduction in paw swelling was seen in rats treated with rAAV5.NFkappaB-TNFRI-Ig. This clinical effect was accompanied by a decrease in pro-inflammatory cytokine levels and an increase in IL10 expression in the synovium. CONCLUSION: These results provide evidence that intra-articular gene therapy using rAAV5 encoding TNFRI-Ig may be a safe and feasible approach for the treatment of rheumatoid arthritis. The higher affinity for human TNFalpha suggests that in patients with rheumatoid arthritis the therapeutic effect might be even more pronounced than in rat adjuvant arthritis.

Intraarticular interferon-beta gene therapy ameliorates adjuvant arthritis in rats.

Interferon (IFN)-beta has significant immunomodulatory properties and has received much interest as a potentially therapeutic agent for rheumatoid arthritis (RA). Systemic IFN-beta treatment of patients with RA was not effective, probably because of pharmacokinetic issues. Therefore, we studied the effect of local IFN-beta production by adenovirus-mediated gene transfer to the ankle joints of arthritic rats. Adjuvant arthritis (AA) in rats was used as a model to study intraarticular gene therapy with an adenoviral vector encoding the rat IFN-beta gene (Ad.IFN-beta). The effect on paw swelling was measured by water displacement plethysmometry. Synovial tissue of the hind paws was examined by immunohistochemistry. Bone destruction was analyzed on the basis of radiographs. In addition, quantitative real-time polymerase chain reaction was used to assess IFN-beta expression. Levels of IFN-beta mRNA and protein peaked 2 days after intraarticular injection and declined thereafter. Local delivery of Ad.IFN-beta after the onset of disease reduced paw swelling significantly. This was accompanied by a reduction in synovial inflammation. The clinical effects in rat AA lasted up to 9 days. Strikingly, Ad.IFN-beta treatment protected bone from erosion, reduced levels of c-Cbl and Cbl-b (both signaling molecules essential for osteoclast activity), and reduced the matrix metalloproteinase-3:tissue inhibitor of metalloproteinase-1 ratio in the joint. Immunohistochemical analysis of the synovial tissue revealed a clear shift toward a more antiinflammatory cytokine profile. Local overexpression of IFN-beta inhibits arthritis progression and protects against bone destruction in rat AA. These findings validate IFN-beta as a therapeutic molecule for intraarticular gene therapy of arthritis.

Gene therapy as a therapeutic approach for the treatment of rheumatoid arthritis: innovative vectors and therapeutic genes.

In recent years, significant progress has been made in the treatment of rheumatoid arthritis (RA). In addition to conventional therapy, novel biologicals targeting tumour necrosis factor-alpha have successfully entered the clinic. However, the majority of the patients still has some actively inflamed joints and some patients suffer from side-effects associated with the high systemic dosages needed to achieve therapeutic levels in the joints. In addition, due to of the short half-life of these proteins there is a need for continuous, multiple injections of the recombinant protein. An alternative approach might be the use of gene transfer to deliver therapeutic genes locally at the site of inflammation. Several viral and non-viral vectors are being used in animal models of RA. The first gene therapy trials for RA have already entered the clinic. New vectors inducing long-term and regulated gene expression in specific tissue are under development, resulting in more efficient gene transfer, for example by using distinct serotypes of viral vectors such as adeno-associated virus. This review gives an overview of some promising vectors used in RA research. Furthermore, several therapeutic genes are discussed that could be used for gene therapy in RA patients.

Enhanced gene transfer to arthritic joints using adeno-associated virus type 5: implications for intra-articular gene therapy.

BACKGROUND: Gene therapy of the joint has great potential as a new therapeutic approach for the treatment of rheumatoid arthritis (RA). The vector chosen is of crucial importance for clinical success. OBJECTIVE: To investigate the tropism and transduction efficiency in arthritic joints in vivo, and in synovial cells in vitro, using five different serotypes of recombinant adeno-associated virus (rAAV) encoding beta-galactosidase or green fluorescent protein genes. METHODS: rAAV was injected into the ankle joints of rats with adjuvant arthritis after the onset of disease. Synovial tissue was examined at different time points for beta-galactosidase protein and gene expression by in situ staining and polymerase chain reaction (PCR) analysis, respectively. In addition, the ability of rAAV to transduce primary human fibroblast-like synoviocytes from patients with RA was investigated in vitro. RESULTS: Intra-articular injection of the rAAV5 serotype resulted in the highest synovial transduction, followed by much lower expression using rAAV2. Expression of the transgene was already detectable 7 days after injection and lasted for at least 4 weeks. Only background staining was seen for serotypes 1, 3, and 4. Importantly, there was a minimal humoral immune response to rAAV5 compared with rAAV2. Additionally, it was found that both rAAV2 and rAAV5 can efficiently transduce human fibroblast-like synoviocytes obtained from patients with RA. CONCLUSION: Intra-articular rAAV mediated gene therapy in RA might be improved by using rAAV5 rather than other serotypes.

Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints.

The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis (RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues. The aim of this study was to compare the transduction efficiency of different AAV serotypes encoding murine secreted alkaline phosphatase (mSEAP) or Escherichia coli beta-galactosidase for intraarticular gene delivery in an experimental model of arthritis. The vectors contained AAV2 terminal repeats flanking the reporter gene in an AAV1, AAV2, or AAV5 capsid, producing the pseudotypes rAAV-2/1, rAAV-2/2, and rAAV-2/5. Left knee joints of mice with collagen-induced arthritis were injected and transgene expression was analyzed by chemiluminescence or direct in situ staining of frozen sections. We show for the first time that intraarticular gene transfer with AAV- 2/5 was far more efficient than with the other serotypes tested. Transgene expression was detectable as early as 7 days after injection, reached a maximum at 21 days, and was stably expressed for at least 130 days, whereas AAV-2/1- and AAV-2/2-mediated expression levels were barely detectable. These findings provide a practical application for future local AAV-mediated gene therapy trials in RA.

Mice lacking the multidrug resistance protein 1 are resistant to Streptococcus pneumoniae-induced pneumonia.

Leukotrienes (LTs) are considered important for antibacterial defense in the lung. Multidrug resistance protein 1 (mrp1) is a transmembrane protein responsible for the cellular extrusion of LTC(4). To determine the role of mrp1 in host defense against pneumonia, mrp1(-/-) and wild-type mice were intranasally inoculated with Streptococcus pneumoniae. mrp1(-/-) mice displayed a diminished outgrowth of pneumococci in lungs and a strongly reduced mortality. These findings were related to an effect of mrp1 on LT metabolism, because survival was similar in mrp1(-/-) and wild-type mice treated with the 5-lipoxygenase-activating protein inhibitor MK-886. Although LTC(4) levels remained low in the bronchoalveolar lavage fluid of mrp1(-/-) mice, LTB(4) concentrations were higher than in wild-type mice. These elevated LTB(4) concentrations were important for the relative protection of mrp1(-/-) mice, because the LTB(4) antagonist LTB(4)-dimethyl amide abolished their survival advantage. In vitro experiments suggested that the intracellullar accumulation of LTC(4) in mrp1(-/-) mice results in product inhibition of LTC(4)-synthase, diminishing substrate competition between LTA(4)-hydrolase (which yields LTB(4)) and LTC(4)-synthase for the available LTA(4). We conclude that mrp1(-/-) mice are resistant against pneumococcal pneumonia by a mechanism that involves increased release of LTB(4). These results identify mrp1 as a novel target for adjunctive therapy in pneumonia.

Depletion of alveolar macrophages exerts protective effects in pulmonary tuberculosis in mice.

Mycobacterium tuberculosis bacilli are intracellular organisms that reside in phagosomes of alveolar macrophages (AMs). To determine the in vivo role of AM depletion in host defense against M. tuberculosis infection, mice with pulmonary tuberculosis induced by intranasal administration of virulent M. tuberculosis were treated intranasally with either liposome-encapsulated dichloromethylene diphosphonate (AM(-) mice), liposomes, or saline (AM(+) mice). AM(-) mice were completely protected against lethality, which was associated with a reduced outgrowth of mycobacteria in lungs and liver, and a polarized production of type 1 cytokines in lung tissue, and by splenocytes stimulated ex vivo. AM(-) mice displayed deficient granuloma formation, but were more capable of attraction and activation of T cells into the lung and had increased numbers of pulmonary polymorphonuclear cells. These data demonstrate that depletion of AMs is protective during pulmonary tuberculosis.

Gene therapy in rheumatic diseases.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint inflammation as well as progressive cartilage and bone destruction. Advances in the understanding of the pathophysiology of RA have led to the development of new therapeutic strategies, including gene therapy. Gene therapy offers a new approach to deliver therapeutic proteins to the joints of arthritis patients. Local as well as systemic gene therapy can be envisaged for the treatment of arthritis. Several viral and non-viral vectors have been used in animal models for rheumatoid arthritis for ex vivo and in vivo delivery of therapeutic genes. Promising pre-clinical data have resulted from the application of these strategies. Using ex vivo gene delivery, successful and safe gene transfer has been demonstrated in the joints of RA patients. Although new insights into the role of cytokines and other mediators of chronic inflammation have provided novel targets for therapeutic intervention, the development of vectors that induce long-term and regulated gene expression remains a challenge.

Half-life of interleukin-1 beta-induced group II phospholipase A2 in rat mesangial cells.

Group II phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. Potent pro-inflammatory cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) have been found to induce sPLA2 synthesis and release from many cell types among which mesangial cells. Although considerable research has been devoted to unravelling the mechanisms underlying the induction of sPLA2 not much is known about the time scale at which the cytokine elicited signals for sPLA2 induction persist in target cells. In this study we addressed that question by using rat renal mesangial cells as a model target cell. We found that after removal of IL-1 beta from the culture medium, the induced-sPLA2 synthesis continues at gradually decreasing rates for approximately 8 h. This is accompanied by a decrease in sPLA2 mRNA levels. Furthermore, with pulse-chase experiments we investigated the half-life of sPLA2 disappearance from the cells. This disappearance was found to be biphasic. A rapidly disappearing pool, constituting approx. 74% of the total, exhibited a half-life of 1.6 +/- 0.2 h. The remaining pool of the induced enzyme was much more stable and its level remained constant for at least 24 h. Analysis of the appearance of newly synthesized enzyme in the culture medium indicated this process to be completed in an hour.

Aspirin inhibits expression of the interleukin-1beta-inducible group II phospholipase A2.

Nonsteroidal anti-inflammatory drugs (NSAIDs) clearly inhibit the synthesis and release of prostaglandins. However, these actions are not sufficient to explain all the anti-inflammatory effects of these drugs. Recently, it has been shown that aspirin and sodium salicylate inhibit the activation of the transcription factor NF-kappaB. Group II phospholipase A2 (sPLA2) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin-1beta (IL-1beta) and this induction is attenuated by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC). We now report that aspirin inhibits the IL-1beta-induced sPLA2 activity in rat mesangial cells in a dose-dependent manner. The IC50 value of aspirin for sPLA2 inhibition was 6.5 mM. This decrease in sPLA2 activity was not due to direct inhibition of enzymatic activity but rather to the fact that aspirin inhibits the expression of IL-1beta-induced sPLA2 protein and mRNA. Furthermore, by electrophoretic mobility shift analysis we demonstrate reduced DNA binding of the nuclear factor kappaB, an essential component of the IL-1beta-dependent upregulation of sPLA2 gene transcription, after treatment of the cells with aspirin. The study described in this report indicates that the inhibition of sPLA2 expression as induced by pro-inflammatory cytokines potentially represents an additional mechanism of action for aspirin.

Effects of dexamethasone and transforming growth factor-beta 2 on group II phospholipase A2 mRNA and activity levels in interleukin 1 beta- and forskolin-stimulated mesangial cells.

The expression of 14 kDa group II phospholipase A2 [also referred to as secretory PLA2 (sPLA2)] is induced in rat glomerular mesangial cells by exposure to inflammatory cytokines and forskolin, a cAMP elevating agent. Previously we have shown that dexamethasone and transforming growth factor-beta 2 (TGF-beta 2) suppress sPLA2 protein synthesis and enzyme activity induced by cytokines and forskolin. The regulation of sPLA2 by pro-inflammatory cytokines suggests that the enzyme may play a role in glomerular inflammatory reactions. In order to understand the regulation of sPLA, in more detail, we investigated whether dexamethasone and TGF-beta 2 also suppress sPLA, mRNA after its induction by either interleukin-1 beta (IL-1 beta) or forskolin. We found that IL-1 beta-induced sPLA2 mRNA in rat mesangial cells is not down-regulated by pretreatment of the cells with dexamethasone, even at a concentration of 10 microM, which dramatically decreases sPLA2 protein levels and activity. Metabolic labelling experiments indicated that the decreased sPLA2 levels under these conditions can be explained by inhibition of the rate of sPLA2 synthesis from the elevated mRNA levels. In contrast, the forskolin-induced elevation of sPLA, mRNA is inhibited by dexamethasone in a concentration-dependent manner. Likewise, TGF-beta 2 inhibits the elevation of sPLA, mRNAs induced by either IL-1 beta or forskolin. The decrease in sPLA2 mRNA caused by TGF-beta 2 corresponds with the decrease in sPLA2 enzyme levels and activity. These data suggest that cytokine- and forskolin-induced sPLA2, expression is tightly controlled via both transcriptional and post-transcriptional mechanisms. Furthermore, we show that pretreatment of mesangial cells with epidermal growth factor prior to stimulation with IL-1 beta or forskolin had no suppressing effect on sPLA2 levels or enzyme activity, as has been reported previously for osteoblasts.

Levels and localization of group II phospholipase A2 and annexin I in interleukin- and dexamethasone-treated rat mesangial cells: evidence against annexin mediation of the dexamethasone-induced inhibition of group II phospholipases A2.

The mechanism by which glucocorticosteroids inhibit the synthesis and secretion of pro-inflammatory arachidonate metabolites is still controversial. Initially it was postulated that glucocorticoids can induce the formation of PLA2 inhibitory proteins termed annexins. We have previously shown that the cytokine-induced 14 kDa PLA2 activity and the synthesis of prostaglandin E2 in rat mesangial cells is dose-dependently blocked by pretreatment of the cells with dexamethasone (Schalkwijk et al. (1991) Biochem. Biophys. Res. Commun. 180, 46-52). Concurrently, the synthesis of 14 kDa group II PLA2 is suppressed. The regulation of PLA2 activity is complex and may well involve superimposable mechanisms. Thus, although the decrease in PLA2 protein levels could in itself explain the dexamethasone-induced decrease in PLA2 activity, a contribution of the glucocorticoid-induced anti-phospholipase A2 protein annexin cannot be ruled out a priori. To investigate this possibility we analyzed the level of annexin I by Western blotting and immunostaining in mesangial cells treated with interleukin-1 beta and/or dexamethasone. Under conditions where 14 kDa group II PLA2 activity and protein levels were dramatically affected by interleukin-1 and dexamethasone, the level of annexin I in the cells remained constant. Dexamethasone also did not induce the secretion of annexin I. In addition, no evidence for dexamethasone-induced translocation of annexin I from the cytosol to membranes, thereby possibly sequestering the substrates for PLA2, was obtained. Immunofluorescence studies localized the cytokine-induced PLA2 to the Golgi area and punctate structures in the cytoplasm. We have also studied the subcellular localization of annexin I in rat mesangial cells using confocal microscopy. These studies located annexin I mainly in the cytoplasma and the nucleus. We conclude from these experiments that the dexamethasone-induced inhibition of 14 kDa group II PLA2 in rat mesangial cells is not mediated by annexin I and is solely due to the suppression of PLA2 gene expression.